Dipyridoxyl phosphate NMRI contrast agents

ABSTRACT

N,N&#39;-bis-(pyridoxal-5-phosphate)-alkylenediamine-N,N&#39;-diacetic acids, N,N&#39;-bis-(pyridoxal-5-phosphate)-1,2-cycloalkylenediamine-N,N&#39;-diacetic acids, and N,N&#39;-bis-(pyridoxal-5-phosphate)-1,2-arylenediamine-N,N&#39;-diacetic acids, the corresponding monophosphate compounds and monoacetic acid compounds, and their salts and esters form stable, highly soluble chelates with paramagnetic metal ions, and are highly effective NMRI contrast agents. Preferred contrast agents are paramagnetic ion chelates of N,N&#39;-bis-(pyridoxal-5-phosphate)ethylene-diamine-N,N&#39;-diacetic acid, N,N&#39;-bis-(pyridoxal-5-phosphate)trans-1,2-cyclohexylenediamine-N,N&#39;-diacetic acid, N,N&#39;-bis-(pyridoxal-5-phosphate)trans-1,2-arylenediamine-N,N&#39;-diacetic acid, and the soluble calcium salts thereof. 
     Novel intermediates for forming these compounds are N,N&#39;-bis(pyridoxal-5-phosphate)alkylenediimines, N,N&#39;-bis(pyridoxal-5-phosphate)alkylenediamines, N,N&#39;-bis(pyridoxal-5-phosphate)-1,2-cycloalkylenediimines, N,N&#39;-bis(pyridoxal-5-phosphate)-1,2-cycloalkylenediamines, N,N&#39;-bis(pyridoxal-5-phosphate(-1,2-arylenediamines, and the corresponding monophosphate compounds.

FIELD OF THE INVENTION

This invention relates to novel compounds which form highly stablechelates with metal ions and which are useful as metal ion carriers forin vivo medical applications. In particular, this invention is directedto novel dipyridoxyl compounds which form highly stable chelates withpolyvalent metal ions, the preparation of the compounds and chelatesthereof with polyvalent ions and particularly paramagnetic ions, and theuse of the paramagnetic chelates as contrast agents in nuclear magneticresonance imagery (NMRI).

BACKGROUND OF THE INVENTION

Traditionally, chelates have been used to administer poorly solublesalts in medicine and as antidotes for detoxification in cases of heavymetal or heavy metal isotope poisoning. Chelates have also been used todeliver radioisotopes to areas of the body for imaging and radiationtherapy. Most recently, chelates with paramagnetic contrast agents havebeen reported for use with NMRI.

Paramagnetic metal ions are frequently toxic in the concentrationsrequired for use in NMRI, and introducing them into the body in the formof chelates renders them more physiologically acceptable. This requiresthat a chelate be able to hold the metal ion tightly in the chelatestructure, that is, the formation constant for the chelate must be verylarge at physiological pH. The paramagnetic metal chelate must also besufficiently soluble to permit administration of quantities required forimaging in reasonable volumes. Usual routes of administration areorally, intravenously and by enema.

The chelating agent must form a stable chelate with those paramagneticmetals which present a hazard to the body if released during use.Paramagnetic metals which are naturally present in the body arepreferred. The chelate forming agent (ligand) must be capable of forminga chelate with a selected paramagnetic material without altering themetal's oxidation state or otherwise reducing its chemical stability.

Since the role of the paramagnetic metal in increasing contrast in NMRIimaging involves reducing the spin-lattice spin relaxation time T₁ andthe spin-spin relaxation time T₂, the chelate structure must hold themetal ion tightly while permitting contact of the metal ion with protonsin water molecules.

This invention provides a novel, superior chelating agent and metalcomplexes therewith which meet the above objectives.

DESCRIPTION OF THE PRIOR ART

A summary of the history and state of the art of contrast agents forNMRI is presented by Valk, J. et al, Basic Principles of NuclearMagnetic Resonance Imaging. New York: Elsevier, pp 109-114 (1985). TheValk et al publication also describes the imaging equipment and methodsfor NMRI, and the entire contents of the Valk et al publication arehereby incorporated by reference in their entirety. Chelates withethylenediaminetetraacetic acid (EDTA) anddiethylaminetriaminepentaacetic acid (DTPA) are described. Toxicityproblems were reduced by seeking less toxic metal ions such as iron andgadolinium in a complex of gadolinium-DTPA chelate-meglumine.Gadolinium, however, is not naturally present in the body and long termtoxicity studies have not been completed. Paramagnetic materials listedin this publication include molecules with unpaired electrons: nitricoxide (NO); nitrogen dioxide (NO₂); and molecular oxygen (O₂). Alsoincluded are ions with unpaired electrons, that is ions from the"transition series". Listed ions include Mn²⁺, Mn³⁺, Fe²⁺, Fe³⁺, Ni²⁺,Cr²⁺, Cu²⁺, the lanthanide series including gadolinium and europium, andnitroxide stable free radicals (NSFR) such as pyrrolidine NSFR andpiperidine NSFR. Toxicity problems are indicated to present a majorproblem with many paramagnetic materials.

Use of alkylenediamine chelates with a variety of paramagnetic ions aredescribed in U.S. Pat. No. 4,647,447. Ferrioxamine-paramagnetic contrastagents are described in U.S. Pat. No. 4,637,929. Manganese(II) is listedas a suitable paramagnetic metal ion for use with polysaccharidederivatives of a variety of chelating compounds including EDTA, DTPA andaminoethyl diphosphonate in PCT application publication no. WO85/05554(Application No. PCT/GB85/00234). Stable radioactive diagnostics agentscontaining ^(99m) Tc chelated with N-pyridoxal-alpha-aminoacids or apyridoxal salt are disclosed in U.S. Pat. Nos. 4,313,928, 4,440,739, and4,489,053.

Taliaferro, C. et al in "New multidentate ligands. 22.N,N'-dipyridoxyethylenediamine-N,N'-diacetic acid: a new chelatingligand for trivalent metal ions", Inorg. Chem. 23:1188-1192 (1984)describe development of N,N,-dipyridoxyethylenediamine-N,N,-diaceticacid (PLED) as a chelating compound for trivalent metal ions. Otherchelating compounds described are the Fe(III) chelates ofN,N'-ethylenebis-2-(o-hydroxyphenyl)glycine (EHPG) andN,N'-bis(1-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED).Properties of chelates of PLED, HBED, EHPG and EDTA with ions of copper,nickel, cobalt, zinc, iron, indium and gallium are compared.Investigation of the structure of PLED is reported by Taliaferro, C. etal, Inorg. Chem. 24:2408-2413 (1985). Green, M. et al, Int. J. Nucl.Med. Biol. 12(5):381-386 (1985) report their evaluation of PLED as achelating ligand for the preparation of gallium and indiumradiopharmaceuticals, and summarize properties of PLED chelates withGa(III), In(III), and Fe(III).

Because the compounds of this invention have an aromatic hydroxy group,their value as chelating agents for manganese(II) ions would not beexpected; such aromatic hydroxy groups would be expected to react withthe manganese(II) ion as an oxidant in the usual way, oxidizing themanganese(II) ion to a higher valence. Frost, et al, J. Am. Chem. Soc.80:530 (1958) report the formation of Mn(II) chelates of EHPG at low pH,but found that attempts to prepare stable manganese(II) complexes withEHPG at higher pH's (above pH 5) was futile as the manganese(II) ion wasirreversibly oxidized. This oxidation occurred even under inertatmospheres, and the writers concluded that the oxidation occurred atthe expense of the ligand or solvent. Anderegg, G. et al, Helv. Chim.Acta. 47:1067 (1964) found the high stability of the Fe(III) chelate ofEHPG was due to the high affinity of the Fe(III) ion for the twophenolate groups present in the ionized ligand. L'Eplathenier, F. et al,J. Am. Chem. Soc. 89:837 (1967) describes studies of HBED involving acidtitrations of HBED in the presence of a variety of metal ions, includingmanganese(II). No manganese chelate was isolated, and the manganeseproducts were not characterized. Based on subsequent work by Patch etal, Inorg. Chem. 21(8):2972-2977 (1982), it is clear that themanganese(II) ion was oxidized by the phenolic ligand during thetitrations of L'Eplathenier et al. Patch et al prepared a Mn(III)complex by reacting Mn(II) salts with EHPG, and concluded the reactioninvolved the oxidation of the ligand in an irreversible reaction. Theability to maintain Mn(III) in the +3 oxidation state was said to be aunique characteristic of the EHPG molecule. U.S. Pat. No. 3,632,637describes phenolic chelating agents such asN,N-di(o-hydroxylbenzyl)-ethylenediamine-N,N'-diacetic acid and theiruse in chelating trivalent and tetravalent metals. These agents areusually stable in the presence of aromatic hydroxy groups. No use of acompound with an aromatic hydroxy group as a chelating agent formanganese(II) ions is disclosed in these references, confirming thegeneral knowledge about the oxidizing properties of the aromatic hydroxygroup on manganese compounds, in particular manganese(II) ions.

SUMMARY OF THE INVENTION

The novel chelate forming compounds of this invention are shown inFormula I. ##STR1## wherein

R is hydrogen or ##STR2##

R₁ is hydrogen or ##STR3## and one of R and R₁ is other than hydrogen;

R₃ is alkylene having from 1 to 8 carbons, 1,2-cycloalkylene having from5 to 8 carbons, or 1,2-arylene having from 6 to 10 carbons, and

R₄ is hydrogen, alkyl having from 1 to 6 carbons or ##STR4##

R₅ and R₆ are each, individually, hydroxy, alkoxy having from 1 to 18carbons, hydroxy-substituted alkoxy having from 1 to 18 carbons, aminoor alkylamido having from 1 to 18 carbons.

The phosphate group mono and diesters with mono and polyhydric alkanolshaving from 1 to 18 carbons, or alkylamino alcohols, each having from 1to 18 carbons, and the salts of the above compounds are included withinthe scope of this invention.

Also included in this invention are the chelates of the compounds ofFormula I and salts and esters thereof with metal ions, preferablyparamagnetic metal ions having atomic numbers of from 21-29, 42, 44 and58-70, and optimally manganese(II), and their use as imaging agents.

The novel intermediate compounds from which the compounds of Formula Iare prepared are also included within the compounds of this invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a structural formula of a species ofN,N'-bis(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid,showing the dissociation constants, pK's, as assigned to the protonationsites described in Example 11.

FIG. 2 is a graph showing the relationship between dosage and relaxivityusing the Mn(DPDP) compound of this invention based on the data shown inExample 9.

DETAILED DESCRIPTION OF THE INVENTION

The novel chelate forming compounds of this invention are shown inFormula I. The pharmaceutically acceptable water-soluble compatiblesalts of the compounds of Formula I and phosphate group esters of thecompounds of Formula I with polyhydric alcohols, aliphatic alcohols, oralkylamino alcohols, each having from 1 to 18 carbons, and the chelatesthereof are also included within the compounds of this invention.

In Formula I, R₅ and R₆ are preferably each individually hydroxy, alkoxyhaving from 1 to 8 carbons, ethylene glycol, glycerol, amino oralkylamido having from 1 to 8 carbons. Optimally, R₅ and R₆ are hydroxyand the salts thereof.

The term "alkyl" and "alkylene", as used herein, include both straightand branch-chained, saturated and unsaturated hydrocarbons. The term"1,2-cycloalkylene" includes both cis and trans cycloalkyl groups andalkyl substituted cycloalkylene groups bonded at the 1,2-positions torespective nitrogen atoms and alkyl substituted derivatives thereofhaving from 5 to 8 carbons. The term "1,2-arylene" includes phenyl andnaphthyl groups bonded at the 1,2-positions to respective nitrogen atomsand alkyl substituted derivatives thereof, having from 6 to 10 carbons.

The compound,N,N'-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N'-diacetic acid orN,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridylmethyl)ethylenediamine-N,N'-diaceticacid, is referred to hereinafter as DPDP, and the Manganese(II) chelateis referred to hereinafter as Mn(DPDP).

The dicarbonyl compound of Formula I, when R₅ and R₆ are hydroxy and R₃is ethylene, is DPDP. DPDP has the dissociation constants for theprotonation sites shown in FIG. 1. As described in Example 11, at pH of3 and above, the ligand is anionic and possesses deprotonated metalbinding sites, both important criteria for metal chelating agents.

CHELATES

The chelates of this invention are chelates of the compounds of FormulaI with metal ions. The chelates can be represented by Formulas II, IIIor IV. ##STR5##

In Formulas II, III and IV, Z represents a metal ion and R₃, R₄, R₅ andR₆ are the same as described with respect to the compounds of Formula I.The dotted lines in the figure represent the dative bonding between theoxygen and nitrogen atoms and the metal ion. One of the acetyl groups inFormula II is below the plane of the aromatic pyridine rings and theother acetyl group is above the plane of the aromatic pyridine rings, sothe metal ion is tightly held within the interior of the chelate saltcomplex with the dicarboxy embodiments of this invention. Also includedin the chelates of this invention are the pharmaceutically acceptablewater-soluble compatible salts, and carboxylic and phosphate groupesters with hydroxy-substituted alkanols, alkanols, or alkylaminoalcohols, each having from 1 to 18 carbons, of the compounds of FormulasII, III, and IV.

For use as a medium for NMRI analysis, the central ion of the complexchelate salt must be paramagnetic, and preferably is a divalent ortrivalent ion of elements with an atomic number of 21 to 29, 42, 44 and58 to 70. Suitable ions include chromium(III), manganese(II), iron(III),iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III),neodymium(III), samarium(III), ytterbium(III). Gadolinium(III),terbium(III), dysprosium(III), holmium(III) and erbium(III) aresometimes preferred because of their strong magnetic moments andchemical stability, but because they are not normally present in thebody, their long term biological effects are unknown.

With the novel chelate forming compounds of Formula I, chelates ofmanganese(II) are preferred. Relatively few manganese(II) chelatecompounds are known, and only a fraction of these have beencharacterized i.e., by single crystal X-ray diffraction. Most of thestructurally characterized Mn(II) complexes have various mono andbidentate ligands coordinating to the metal center. The Mn(II) complexesof Formulas II, II and III, and the Mn(II) complexes with PLED and thecorresponding 1,2-cycloalkylene and 1,2-arylene compounds described inour co-pending, concurrently filed application U.S. Ser. No. 47,584filed May 8, 1987 titled Manganese(II) Chelate Contrast Agents andMethods are the first Mn(II) complexes with a high affinity hexadentateligand. This configuration provides a more stable and effective form forintroducing manganese(II) into the body as a NMRI contrast medium.

The manganese(II) complex of Formula II (R₅ and R₆ =hydroxy, R₃=ethylene), Mn(DPDP), was studied potentiometrically from pH 11.1 to2.0. The data obtained was analyzed using a model consisting of threeone proton steps and one two proton step. Refinement yielded equilibriumconstants with calculated e.s.d.'s (log K) of less than 0.02. The logK_(f) (formation constant) for Mn(DPDP) was calculated to be 14.8 and isidentical to that of the manganese chelate ofN,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED)reported by L'Eplathenier, F. et al (supra). The titration dataindicates that Mn(DPDP) begins to demetallate as the pH drops below 4.5.At physiological pH, however, the Mn(II) ion is quantitatively bound tothe chelating agent, stabilized at the +2 valence.

The chelate forming compounds of this invention are also suitable forforming chelate salts of other metals intended for X-ray diagnosis. Ingeneral, these are the elements of an atomic number which issufficiently high to efficiently absorb X-rays. Diagnostic mediacontaining a physiologically well tolerated chelate salt with centralions of elements with atomic numbers of 57 to 83 are suitable for thispurpose. Included in this group are lanthanum(III), gold(III), lead(II),and bismuth(III).

All of the chelates according to this invention useful for NMRI andX-ray analysis are also suitable for use in ultrasonic diagnosis.

The elements of the above-listed atomic numbers which form the centralion or ions of the physiologically well tolerated chelate salt, must notbe radioactive for the intended use of the diagnostic medium for X-raydiagnosis and NMRI. Radioactive metal chelates of the compounds ofFormula I are described in our U.S. Ser. No. 47,616 filed May 8, 1987,and U.S. Pat. No. 4,842,845, issued June 27, 1989 titled DipyridoxylPhosphate Radioactive Metal Chelates.

For purposes of clarity, the chelates of this invention will bedescribed hereinafter in terms of paramagnetic ions suitable for use inNMRI analysis. However, this is for purposes of clarity of explanationand not by way of limitation, and chelates of all of the above metalions are included within the scope of this invention.

If not all of the active hydrogen atoms of the chelates are substitutedby the central paramagnetic ion, the solubility of the chelate isincreased if the remaining hydrogen atoms are substituted withphysiologically biocompatible cations of inorganic and/or organic basesor amino acids. For example, the lithium ion, the potassium ion, thesodium ion and especially the calcium ion are suitable inorganiccations. Suitable cations of organic bases include, for example,ethanolamine, diethanolamine, morpholine, glucamine,N,N-dimethylglucamine, and N-methylglucamine. Lysine, arginine ororithine are suitable as cations of amino acids, as generally are thoseof other basic naturally occurring acids.

The preferred calcium salts have calcium ion to chelating molecule moleratios of from 0.05 to 1.0, the optimum mole ratios being with the rangeof from 0.1 to 0.5. At mole ratios of calcium ion to chelate moleculeabove 1.0, the chelate tends to become insoluble. The soluble calciumsalts are most physiologically acceptable since they do notsignificantly disturb the concentration of free calcium ions in thepatient's system.

The chelates according to this invention are formed from the chelateforming compounds of Formula I by conventional procedures known in theart. In general, these processes involve dissolving or suspending themetal oxide or metal salt (for example, nitrate, chloride or sulfate) ofan element with an atomic number of 21 to 29, 42, 44 or 57 to 83 (forexample, oxides or salts of Mn⁺², Cr⁺³, Fe⁺², Fe⁺³, Co⁺³, Ni⁺², Cu⁺²,Pr⁺³, Nd⁺³, Sm⁺³, Yb⁺³, Gd⁺³, Tb⁺³, Dy⁺³, Ho⁺³, or Er⁺³) in water or alower alcohol such as methanol, ethanol or isopropanol. To this solutionor suspension is added an equimolar amount of the chelating acid inwater or a lower alcohol, and the mixture is stirred, if necessary, withheating moderately or to the boiling point, until the reaction iscompleted. If the chelate salt formed is insoluble in the solvent used,the reaction product is isolated by filtering. If it is soluble, thereaction product is isolated by evaporating the solvent to dryness, forexample, by spray drying or lyophilizing.

If acid groups such as the phosphoric acid groups are still present inthe resulting chelate, it is advantageous to convert the acidic chelatesalt into a neutral chelate salt by reaction with inorganic and/ororganic bases or amino acids, which form physiologically biocompatiblecations, and to isolate them. This is often unavoidable since thedissociation of the chelate salt is moved toward neutrality to such anextent by a shift in the pH value during the preparation that only inthis way is the isolation of homogeneous products or at least theirpurification made possible. Production is advantageously performed withorganic bases or basic amino acids. It can also be advantageous,however, to perform the neutralization by means of inorganic bases(hydroxides, carbonates or bicarbonates) of sodium, potassium orlithium.

To produce the neutral salts, enough of the desired base can be added tothe acid chelate salts in an aqueous solution or suspension that thepoint of neutrality is reached. The resulting solution can then beconcentrated to dryness in vacuo. It is often advantageous toprecipitate the neutral salts by adding a solvent miscible with water,for example, lower alcohols (methyl, ethyl, isopropyl alcohols, etc.),lower ketones (acetone, etc.), polar ethers (tetrahydrofuran,1,2-dimethoxyethane, etc.) and thus obtain crystals that isolate easilyand purify well. It has been found particularly advantageous to add thedesired bases to the reaction mixture even during chelating and thuseliminate a process stage. Other conventional purification proceduressuch as column chromatography can be used.

Since the chelate salts of Formulas II, III and IV contain a pluralityof acid groups, it is possible to produce neutral mixed salts whichcontain both inorganic and organic physiologically biocompatible cationsas counterions. This can be done, for example, by reacting thecomplexing acids in an aqueous suspension or solution with the oxide orsalt of the element supplying the central ion or less than the fullamount of an organic base necessary for neutralization, e.g., half,isolating the chelate salt that is formed, purifying it, if desired, andthen adding it to the amount of inorganic base necessary for completeneutralization. The sequence of adding the bases can be reversed.

The carboxylic and phosphoric acid groups of the chelating agents canalso be neutralized by esterification to prepare carboxylate andphosphate esters. Such esters can be prepared by conventional proceduresknown in the art, for example, from the corresponding alcohols. Suitableesters include, for example, esters of straight or branch-chainedalkanol groups having from 1 to 18 carbons, mono and polyhydric alkylamino alcohols having from 1 to 18 carbons and preferably from 1 to 6carbons such as serinol or diethanolamine, and polyhydric alcoholshaving from 1 to 18 and preferably from 1 to 6 carbons such as ethyleneglycol or glycerol.

The diagnostic media for administration is formed using physiologicallyacceptable media in a manner fully within the skill of the art. Forexample, the chelate salts, optionally with the addition ofpharmaceutically acceptable excipients, are suspended or dissolved in anaqueous medium, and then the solution or suspension is sterilized.Suitable additives include, for example, physiologically biocompatiblebuffers (as, for example, tromethamine hydrochloride), slight additionsof other chelating agents (as for example, diethylenetriaminepentaceticacid) or, optimally, calcium salts (for example, calcium chloride,calcium ascorbate, calcium gluconate or calcium lactate).

Alternatively, the diagnostic media according to this invention can beproduced without isolating the chelate salts. In this case, special caremust be taken to perform the chelating so that the salts and saltsolutions according to the invention are essentially free of unchelated,potentially toxic metal ions. This can be assured, for example, usingcolor indicators such as xylenol orange to control titrations during theproduction process. A purification of the isolated salt chelate can alsobe employed as a final safety measure.

If suspensions of the chelate salts in water or physiological saltsolutions are desired for oral administration, a small amount of solublechelate salt can be mixed with one or more of the inactive ingredientstraditionally present in oral solutions such as surfactants, aromaticsfor flavoring and the like.

The most preferred mode for administering paramagnetic metal chelates ascontrast agents for NMRI analysis is by intravenous administration.Intraveneous solutions must be sterile, free from physiologicallyunacceptable agents, and should be isotonic or iso-osmotic to minimizeirritation or other adverse effects upon administration. Suitablevehicles are aqueous vehicles customarily used for administeringparenteral solutions such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, and other solutions such as are describedin Remington's Pharmaceutical Sciences. 15th Ed., Easton: MackPublishing Co. pp 1405-1412 and 1461-1487 (1975) and The NationalFormulary XIV. 14th Ed. Washington: American Pharmaceutical Association(1975), the contents of which are hereby incorporated by reference. Thesolutions can contain preservatives, antimicrobial agents, buffers andantioxidants conventionally used in parenteral solutions, selectingexcipients and other additives which are compatible with the chelatesand which will not interfere with the manufacture, storage or use of theproducts.

The diagnostic media according to this invention can contain from 0.001to 5.0 moles per liter and preferably from 0.1 to 0.5 moles per liter ofthe chelate salt.

The chelates of this invention are administered to patients for imagingin amounts which are sufficient to yield the desired contrast.Generally, dosages of from 0.001 to 5.0 mmoles of contrast agent perkilogram of patient body weight are effective to achieve reduction ofrelaxivity rates. The preferred dosages for most NMRI applications arefrom 0.02 to 0.5 mmoles of contrast agent per kilogram of patient bodyweight.

Methods for applying the contrast agents to improve NMRI images,equipment and operating procedures are described by Valk, J. et al,supra. The contrast agents can be used orally and intravenously.

In a novel NMRI application, the contrast agents can be introduced intothe cervix, uterus and fallopian tubes. NMR imaging can then beperformed to detect causes of infertility such as obstructions orimperfections in the internal surface of the fallopian tubes which mightinterfere with the movement of the fertilized ovum.

CHELATE FORMING COMPOUNDS

The compounds of Formula I can be formed by reacting the correspondingpyridoxal 5-phosphate(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridinecarboxyaldehyde)represented by Formula V with a diamine represented by Formula VI.##STR6## In the compounds of Formula V and VI, R₃ and R₄ are as definedwith respect to Formula I. Pyridoxyl 5-phosphate, pyridoxal, and theother compounds of Formula V, and the alkylenediamine,cycloalkylenediamine and arylene reactants of Formula VI are well knowncompounds readily available from commercial sources, and they can bereadily synthesized by well known procedures fully within the skill ofthe art.

The reaction of the amino groups of the compounds of Formula VI with thealdehyde group of the compounds of Formula V can be carried out in analcohol such as methanol at a temperature within the range of from 0° to60° C. The diimines formed are represented by Formula VII. ##STR7## Inthe compounds of Formula VII, R₃ and R₄ are the same as described withrespect to the compounds of Formula I. For the manufacture of compoundswherein R₄ is a phosphonomethyl group, i.e., the5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)-methylideneaminoalkyleneiminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoricacids,5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)-methylideneamino-1,2-cycloalkyleneiminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoric acids, and5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)-methylideneamino-1,2-aryleneiminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoric acids of Formula VII, a diamine of Formula VI is reacted withtwo molar equivalents of an aldehyde of Formula V having the5-phosphonomethyl group such as pyridoxyl 5-phosphate. For preparationof compounds of Formula VII wherein R₄ is other than a phosphonomethylgroup, the diamine of Formula VI is first reacted with only one molarequivalent of an aldehyde of Formula V having the 5-phosphonomethylgroup, and the mono-phosphonomethyl reaction product is reacted with onemolar equivalent of a compound of Formula V having the desired R₄ group,such as a 5-hydroxymethyl group, i.e., pyridoxal. The reverse order ofreaction can also be used. The reaction products of Formula VII areinsoluble in the alcohol and can be isolated by filtration.

The compounds of Formula VII are then hydrogenated by conventionalprocedures using a palladium or platinum catalyst to yield the diaminesof Formula VIII. ##STR8## In the compounds of Formula VIII, R₃ and R₄are the same as described with respect to the compounds of Formula IV.The5-(N-(3-hydroxy-2-methyl-5-phospono-methyl-4-pyridyl)-methylaminoalkyleneaminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoricacids,5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)-methylamino-1,2cycloalkyleneaminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoric acids,5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)methylamino-1,2-cycloaryleneaminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoric acids, and the monophosphonomethyl compounds of Formula VIIIcan be left in solution or isolated as crystalline solids.

The compounds of Formula I are prepared by reacting the diamines ofFormula VIII with a haloacetic acid such as bromoacetic acid, the molarratio of the bromoacetic acid to diamine determining whether one or bothof the amines are conjugated with the acetic acid groups. TheN,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridyl-methyl)alkylenediamine-N,N'-diaceticacids,N,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridyl-methyl)-1,2-cycloalkylenediamine-N,N'-diaceticacids,N,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridyl-methyl)-1,2-arylenediamine-N,N'-diaceticacids,N,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridylmethyl)alkylenediamine-N-aceticacids,N,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridyl-methyl)-1,2-cycloalkylenediamine-N-aceticacids, andN,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridyl-methyl)-1,2-arylenediamine-N-aceticacids of Formula I are then isolated and purified by conventionalprocedures such as recrystallization or anion exchange chromatography.

The carboxylic acid esters and amides can be formed by conventionalprocedures reacting the carboxylic acids with alkanols having from 1 to18 carbons, hydroxy-substituted alkanols having from 1 to 18 carbons,ammonia, and alkylamines having from 1 to 18 carbons.

This invention is further illustrated by the following specific butnon-limiting examples. Temperatures are given in degrees centigrade andconcentrations as weight percents unless otherwise specified. Procedureswhich are constructively reduced to practice herein are described in thepresent tense, and procedures which have been carried out in thelaboratory are set forth in the past tense.

EXAMPLE 1 N,N'-bis(pyridoxal-5-phosphate)ethylenediimine

A 265.2 gm (1 mole) quantity of pyridoxal-5-phosphate (Chemical DynamicsCorp., South Plainfield, N.J.) was slurried in one liter of methanol,and 400 ml of 5M NaOH was added. When the solution was homogeneous, 34.2ml of 1,2-diaminoethane (Aldrich Chem. Co.) was added rapidly withvigorous stirring. The imine product sodiumN,N'-bis(pyridoxal-5-phosphate)ethylenediimine or sodium5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)methylideneaminoethyleneiminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphatewas stirred for 1 hr, 400 ml of diethyl ether was added, and the slurrywas filtered. The filtrate was washed with 600 ml of ethanol and driedat 60° C. in vacuo. A 290 gm quantity of the bis-imine with a meltingpoint of 215°-220° C. (decomposition) was isolated (90% yield, based onthe tetra-sodium salt). IR (KBr) pellet: 1630 cm⁻¹ (C═N), ¹ H NMR (D₂ O,400 MHz) delta 8.88 (s, --N═CH), 7.54 (s, pyr-H), 4.70 (d, CH₂ OP,J_(HP) =6.3 Hz), 4.06 (s, NC₂ CH₂ N), 2.21 (s, pyr-CH₃).

EXAMPLE 2 N,N'-bis(pyridoxal-5-phosphate)alkyldiimines

Repeating the procedure of Example 1 but replacing the 1,2-diaminoethanewith 1,3-diamino-n-propane, 1,2-diamino-n-propane,1,2-diaminoisopropane, 1,2-diamino-n-butane, 1,4-diamino-n-butane,1,3-diamino-n-butane, 1,2-diamino-3-methylpropane yields thecorresponding N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-propylene)diimine,N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-propylene)diimine,N,N'-bis(pyridoxal-5-phosphate)-1,2-isopropylenediimine,N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-butylene)diimine,N,N'-bis(pyridoxal-5-phosphate)-1,4-(n-butylene)diimine,N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-butylene)diimine, andN,N'-bis(pyridoxal-5-phosphate)-1,2-(3-methyl)propylenediimine.

EXAMPLE 3 N,N'-bis(pyridoxal-5-phosphate)ethylenediamine

The diimine from Example 1 was charged to a 5 liter 3-neck flask fittedwith mechanical stirrer, fritted tube bubbler, and a 3-way stopcock.Then 1.5 liters of deionized water was added, followed by 1.5 liters ofmethanol. The yellow solution formed was stirred while sparging withnitrogen. Then 13 gm of 5% Pt on carbon (Aldrich Chem. Co.) was added,and the apparatus was purged with hydrogen. The reaction was allowed toproceed for 5 hr with continuous addition of hydrogen. HPLC analysisshowed complete reduction to the amine. The reaction mixture was spargedwith nitrogen for 15 min and then filtered through Celite. The filtratewas concentrated in vacuo at 60° C. to about 500 ml. The solution,containing N,N'-bis(pyridoxal-5-phosphate)ethylenediamine or5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)methylaminoethyleneaminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoricacid salt was used directly for the next step. If desired the diaminecan be isolated as off-white crystals by the addition of 200 ml of 97%formic acid and allowing the product to crystallize at room temperatureovernight. The diamine is isolated by filtration and washed with 2×150ml of cold deionized water. ¹ H NMR (D₂ O, 400 MHz) delta 7.47 (s,pyr-H), 4.58 (d, CH₂ OP, J_(HP) =6.3 Hz), 3.94 (s, NCH₂ CH₂ N), 2.88 (s,N-CH₂ -pyr), 2.16 (s, pyr-CH₃).

EXAMPLE 4 N,N'-bis(pyridoxal-5-phosphate)alkyldiamines

Repeating the procedure of Example 3 but substituting the diimineproducts of Example 2 for the diimine product of Example 1 yieldsN,N'-bis(pyridoxal-5-phosphate)-1,3-(n-propylene)diamine,N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-propylene)diamine,N,N'-bis(pyridoxal-5-phosphate)-1,2-isopropylenediamine,N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-butylene)diamine,N,N'-bis(pyridoxal-5-phosphate)-1,4-(n-butylene)diamine,N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-butylene)diamine, andN,N'-bis(pyridoxal-5-phosphate)-1,2-(3-methyl)propylenediamine.

EXAMPLE 5 DPDP Synthesis

The diamine from Example 3 was charged to a two liter 4-neck flaskequipped with two addition funnels, pH electrode, thermometer and stirbar. A 100 gm (2.5 mole) quantity of NaOH was dissolved in 200 ml ofdeionized water, and 130 gm (0.9 mole) of bromoacetic acid (Sigma Chem.Co.) was dissolved in 180 ml of deionized water. Each solution wascharged to an addition funnel. Enough NaOH solution was added to thediamine solution to bring the pH to 11. The temperature of the reactionwas raised to 42° C., and bromoacetic acid and NaOH solution were addedconcurrently to maintain the pH at 11. The addition was stopped at 45min, and the progress of the reaction was checked by HPLC. The additionof bromoacetic acid and NaOH was resumed, and the reaction checked at 60and 75 min. All the bromoacetic acid had been added, and the reactionwas complete by HPLC analysis. Approximately 30 ml of the 50% NaOHsolution remained in the addition funnel. A 675 gm quantity of cationexchange resin (AMBERLITE IRC-50) was added, and the mixture was placedin a refrigerator for 14 hr. The pH had dropped to 6.5. The resin wasremoved by filtration, and the filtrate treated with 260 gm of cationexchange resin (DOWEX 50W-X8). The pH dropped to about 4. The resin wasremoved by filtration, and the solution was concentrated in vacuo at 60°C. to a viscous oil. The oil was dried in vacuo for 48 hr at 25° C. toyield a resinous solid containingN,N'-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid orN,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridylmethyl)ethylenediamine-N,N'-diaceticacid (DPDP).

EXAMPLE 6 DPDP Purification

The resinous solid obtained in Example 5 was dissolved in 600 ml of 88%formic acid, 1.5 liters of methanol followed by 2.2 liters of ethanolwas added, and the mixture was cooled to 0° C. for 2 hr. The solventmixture was decanted from the resulting gum and discarded. The gum wasdissolved in about 800 ml of deionized water which was then concentratedin vacuo to about 600-650 ml. Seed crystals were added, and the solutionwas allowed to stand at rm temp overnight. The product was isolated byfiltration, washed with about 400 ml of cold deionized water, 250 ml ofethanol, and then dried in vacuo to yield 65 gm of DPDP in 85-90% purityby HPLC. The filtrate and washings were retained, concentrated in vacuoto about 350 ml, and the solution refrigerated until columnchromatographic purification of the second crop.

The 65 gms of product was then dissolved in 75 ml of 88% formic acidcontaining 5 ml of deionized water with gentle heating to about 60° C.Cold deionized water was added to a total volume of one liter, and thesolution was allowed to stand at 25° C. for 16 hr to crystallize. Theproduct was isolated by filtration, washed with 200 ml cold deionizedwater, and dried in vacuo at 60° C. to yield 55 gms of DPDP in 93-95%purity by HPLC. A second recrystallization, using the same procedureyields 50 gm of DPDP in 96-98% purity by HPLC, mp 174°-180° C. withdecomposition. Analysis: (Calculated for C₂₂ H₃₂ N₄ O₁₄ P₂) C, 41.38; H,5.05; N, 8.77. (Found) C, 40.70; H, 5.14; N, 8.61. ¹ H NMR (D₂ O, 400MHz) delta 7.93 (s, pyr-H), 4.81 (d, CH₂ OP, J_(HP) =6.3 Hz), 4.07 (s,NCH₂ CH₂ N), 3.35 (s, CH₂ COOH), 2.83 (s, N-CH₂ -pyr), 2.38 (s,pyr-CH₃). ³¹ P NMR (D₂ O, 161 MHz) delta -1.61 (s, CH₂ OP, H₃ PO₄reference).

EXAMPLE 7 Sodium-Calcium Salt of Mn(DPDP)

A 4.16 gm (6.25 mmole) portion of DPDP from Example 6 was dissolved in15 ml of rigorously degassed water by the addition of 1.0 gm (25 mmoles)of NaOH. A 1.25 gm (6.25 mmole) quantity of manganese dichloridetetrahydrate was added, and the solution immediately turned yellow.After stirring for 30 min, 0.25 gm (6.25 mmole) of solid NaOH was addedto bring the pH up to 6.5. Then 0.15 gm (1.0 mmole) of calcium chloridewas added, and sufficient degassed water was added to bring the volumeof the solution to 25 ml. The clear yellow solution was sterilized bybeing filtered through a 0.2 micron filter to yield the sodium-calciumsalt of a manganese chelate complex ofN,N,-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid orN,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridylmethyl)ethylenediamine-N,N'-diaceticacid.

EXAMPLE 8 Relaxivities with Mn(DPDP)

The relations of protons present in water and plasma exposed to thechelate product of Example 7 was tested by NMR for relaxities, in msec,at 10 MHz, 37° C. The results are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Relaxivities, msec.                                                           Molar    T.sub.1  T.sub.2    T.sub.1                                                                              T.sub.2                                   Conc.    (Water)  (Water)    (Plasma)                                                                             (Plasma)                                  ______________________________________                                        0.010     43      41          40    34                                        0.005    100      86          74    68                                        0.0025   175                 139    124                                       0.00125  332                 240                                              0.000625 639                 398                                              0.000312 1083                624                                              0.000156 1470                856                                              0.000078                     995                                              0.000039                     1103                                             ______________________________________                                    

EXAMPLE 9 Organ Distribution of Mn(DPDP) in Rabbits

Each of four rabbits was injected intravenously with one of thefollowing amounts of the solution obtained in Example 7: 0.01 mmoles/kg,0.05 mmoles/kg, 0.10 mmoles/kg and 0.20 mmoles/kg. The rabbits weresacrificed 30 min post injection, and the proton relaxation values ofselected body organs were measured with NMR, in vitro at 10 MHz. Therelaxation rates found are shown in Table II.

                                      TABLE II                                    __________________________________________________________________________    Relaxivities. msec.                                                                      Observed Values                                                    Normal     Dose (mmol/kg)                                                     Values     0.01  0.05   0.10  0.20                                            Tissue                                                                            T.sub.1                                                                          T.sub.2                                                                           T.sub.1                                                                          T.sub.2                                                                          T.sub.1                                                                          T.sub.2                                                                           T.sub.1                                                                          T.sub.2                                                                          T.sub.1                                                                          T.sub.2                                      __________________________________________________________________________    Brain                                                                             -- --  554                                                                              76 496                                                                              82  590                                                                              90 352                                                                              66                                           Heart                                                                             605                                                                              70  -- -- 353                                                                              54  300                                                                              51 205                                                                              47                                           Lung                                                                              595                                                                              112 -- -- 575                                                                              113 435                                                                              61 376                                                                              63                                           Fat 171                                                                              154 -- -- 200                                                                              139 183                                                                              115                                                                              192                                                                              --                                           Skel.                                                                             423                                                                              47  -- -- 494                                                                              47  425                                                                              31 232                                                                              31                                           Musc.                                                                         Renal                                                                             338                                                                              85  298                                                                              65 210                                                                              57  188                                                                              55 143                                                                              53                                           Cort.                                                                         Renal                                                                             672                                                                              149 502                                                                              99 223                                                                              57  209                                                                              48 127                                                                              60                                           Med.                                                                          Liver                                                                             252                                                                              64  176                                                                              39  76                                                                              31   65                                                                              23  68                                                                              25                                           Stom.                                                                             349                                                                              69  -- -- 245                                                                              41  242                                                                              52 271                                                                              53                                           Small                                                                             352                                                                              79  324                                                                              72 237                                                                              52  218                                                                              46 131                                                                              46                                           Int.                                                                          Large                                                                             349                                                                              77  283                                                                              64 365                                                                              83  290                                                                              57 256                                                                              74                                           Int.                                                                          Urine                                                                             -- --  821                                                                              -- 150                                                                              136  90                                                                              75 -- --                                           Blood                                                                             900                                                                              --  844                                                                              -- 613                                                                              --  506                                                                              -- 411                                                                              --                                           __________________________________________________________________________

The organ distribution data is plotted in FIG. 2, with the followingsymbols:

    ______________________________________                                        Liver                                                                         1-                 Heart                                                      h-                                Cortex                                      c-                                                                            Medula                                                                        m-        Urine                                                               u-        Blood                                                               b-                                                                            ______________________________________                                    

It shows rapid uptake of the Mn(DPDP) in the heart, liver and kidneys.The liver and kidneys are saturated with a dose of 0.10 mmole/kg whilethe heart continues to uptake Mn(DPDP) through the dose range studied.The complex of Mn(DPDP) may cross the intake-brain barrier as uptake bythe brain was observed at higher doses. In cases where a defect ispresent in the blood-brain barrier (through disease or trauma), largeamounts of the complex of Mn(DPDP) collect in the extravascular spaceand such defects were observed by NMRI tomography. The same defects arenot observable without the use of Mn(DPDP) as a contrast agent.

EXAMPLE 10 Pharmacokinetics with Mn(DPDP)

Each of seven rabbits was injected intravenously with 0.01 mmol/kg ofthe solution obtained in Example 7. The rabbits were sacrificed at 0.25,0.50, 1.0, 2.0, 4, 6 and 24 hours post-injection, and the protonrelation values of selected body organs were measured with NMR, invitro, at 10 MHz. The T₁ relaxation rates are shown in Table III.

                                      TABLE III                                   __________________________________________________________________________    T.sub.1 Relaxivities, msec                                                    Time, hr                      T.sub.1 Normal                                  Tissue                                                                              0.25                                                                             0.50                                                                              1.0                                                                              2   4  6   24 Value                                           __________________________________________________________________________    Liver  68                                                                               76  48                                                                               48 145                                                                              209 315                                                                              250 ± 50                                     Bile  160                                                                               46  25                                                                               21 <1  14 107                                                                              275 ± 55                                     Renal 202                                                                              191 229                                                                              192 210                                                                              239 328                                                                              338 ± 60                                     cortex                                                                        Renal 192                                                                              231 236                                                                              310 340                                                                              375 563                                                                               672 ± 100                                   medulla                                                                       Heart 231                                                                              352 381                                                                              507 522                                                                              663 660                                                                               605 ± 100                                   __________________________________________________________________________

The pharmacokinetic data show rapid uptake and clearance of Mn(DPDP) inthe liver, renal cortex, renal medulla and heart. The results indicateclearance of Mn(DPDP) through both the renal and hepatobiliary systemswithin 6-8 hours post-injection.

EXAMPLE 11 Potentiometric Titrations

The compound DPDP was studied potentiometrically from pH 11.2 to 2.0.Data sets were collected on a custom-built automatic potentiometrictitration apparatus composed of a METROHM 655 DOSIMAT automatic buret, aFISHER ACCUMET pH meter with a CORNING calomel combination electrode, acustom-blown water jacketed titration cell, a BRINKMAN LAUDA K-2/Rconstant temperature bath and a COMMODORE 64 computer. The BASICcomputer program TITRATOR (Harris, W. et al, J. Am. Chem. Soc. 101:6534(1979)) runs the apparatus. Data analysis was performed on an IBM-ATcomputer using the least squares program, BETA (Harris, W. et al,supra), and the data analysis program, HANDNBAR (Harris, W. et al,supra). The titrants were standardized by phenophthalein titration asfollows: KOH was calibrated against potassium hydrogen phthalate (aprimary standard), and HCl solutions were calibrated against the KOHstandard. The Mn(II)Cl₂ solution was standardized with an EDTA titrationusing Erichrome Black T as the indicator. All solutions were made fromdistilled, deionized water that was further purified on a MILLI-Qcartridge system, degassed, and then kept under an atmosphere of argonwhich had been scrubbed for CO₂ and O₂. Additions of EDTA and Mn(II)solutions were performed using calibrated GILMOT pipets. The electrodewas calibrated in concentration units with degassed solutions ofp[H+]=2.291 and 1.078 at 0.1M ionic strength.

The ligand proton titration was performed by adding 28.7 mg (0.045mmoles) to 54.6 ml of a high pH aqueous solution. It was then titratedto low pH with 0.1009N HCl.

The metal complex titration was performed by adding 152.2 mg (0.2383mmoles) to 74.6 ml of a high pH aqueous solution. 2.07 ml of a 0.1152MMn(II) solution (0.2386 mmoles) were added. The complex was thentitrated to low pH with 1.002N HCl.

The data obtained was analyzed using a model that consisted of eightone-proton steps. The first two protonation equilibria were outside ofthe range of the titration window afforded by the concentration oftitrant and were therefore estimated based on work by Martell andco-workers reported by Taliaferro, C. et al, Inorg. Chem. 24:2408-2413(1985). Refinement of the remaining equilibria yielded constants withcalculated e.s.d.'s (log K) of less than 0.02. Assignment of theprotonation sites (pK's) was based on work by Martell and co-workers(Taliaferro, et al, supra), and is shown in FIG. 1.

At a pH of 3 and above, the ligand is anionic and possesses deprotonatedmetal binding sites, both important criteria for a metal chelatingagent.

EXAMPLE 12 N,N'-bis-(pyridoxal-5-phosphate)alkylenediamine-N,N'-diaceticacids

Repeating the procedure of Examples 5 and 6 but replacing the diamine ofExample 3 with the products of Example 4 yields

N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-propylene)-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-propylene)-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-isopropylene-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-butylene)-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,4-(n-butylene)-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-butylene)-N,N'-diacetic acid, and

N,N'-bis(pyridoxal-5-phosphate)-1,2-(3-methylene)propyl-N,N'-diaceticacid.

EXAMPLE 13 DPDP Chelates

Repeating the procedure of Example 7 but replacing manganese dichloridetetrahydrate with equimolar amounts of the soluble chlorides of Cr⁺³,Fe⁺², Fe⁺³, Co⁺³, Ni⁺², Cu⁺², Pr⁺³, Nd⁺³, Sm⁺³, Yb⁺³, Gd⁺³, Tb⁺³, Ho⁺³,or Er⁺³ yields the corresponding sodium salts of the respective metalion chelates ofN,N'-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid. Theprocedure can be repeated replacing the metal chloride salts withsoluble nitrate or sulfate salts.

EXAMPLE 14 Other Chelates

Repeating the procedure of Example 7 but replacingN,N'-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid withequimolar amounts of the chelate forming compounds produced inaccordance with Example 10, and replacing manganese dichloridetetrahydrate with equimolar amounts of the soluble chlorides, carbonatesor nitrates of Cr⁺³, Fe⁺², Fe⁺³, Co⁺³, Ni⁺², Cu⁺², Pr⁺³, Nd⁺³, Sm⁺³,Yb⁺³, Gd⁺³, Tb⁺³, Ho⁺³, or Er⁺³ yields the sodium-calcium salts of therespective metal ion chelates of

N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-propylene)-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-propylene)-N,N'-diacetic acid,

N,n'-bis(pyridoxal-5-phosphate)-1,2-isopropylene-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-butylene)-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,4-(n-butylene)-N,N'-diacetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-butylene)-N,N'-diacetic acid, and

N,N'-bis(pyridoxal-5-phosphate)-1,2-(3-methylene)propyl-N,N'diaceticacid.

EXAMPLE 15 N,N'-bis(pyridoxal-5-phosphate)trans-1,2-cyclohexylenediimine

A 26.5 gm quantity (0.1 mole) of pyridoxal-5-phosphate was dissolved in300 ml of methanol, and 38 ml of 5N NaOH was added. Then 5.71 gm (0.05mole) of trans-1,2-diaminocyclohexane was added with stirring, and thevolume of the solution was reduced to 200 ml in vacuo. After cooling to0° C., the yellow imine was isolated by filtration, washed with diethylether, and dried in vacuo to yield 17 gm of sodiumN,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclohexylenediimine or sodium5-(N-(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridyl)methylideneamino-trans-1,2-cyclohexyleneiminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphate(49% yield, melting point 200°-205° C. with decomposition).

EXAMPLE 16 Other N,N'-bis(pyridoxal-5-phosphate)-1,2-cyclo(alkylene orarylene)diimines

Repeating the procedure of Example 15 but replacing thetrans-1,2-diaminocyclohexane with trans-1,2-diaminocyclopentane,trans-1,2-diaminocycloheptane, trans-1,2-diaminocyclooctane,cis-1,2-diaminocyclohexane, trans-1,3-diaminocyclohexane,trans-1,4-diaminocyclohexane, o-aminoaniline andcis-1,4-diaminocyclohexane yields the corresponding

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclopentylenediimine,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cycloheptylenediimine,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclooctylenediimine,

N,N'-bis(pyridoxal-5-phosphate)-cis-1,2-cyclohexylenediimine,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,3-cyclohexylenediimine,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,4-cyclohexylenediimine,

N,N'-bis(pyridoxal-5-phosphate)-1,2-phenylenediimine, and

N,N'-bis(pyridoxal-5-phosphate)-cis-1,4-cyclohexylenediimine.

EXAMPLE 17 N,N'-bis(pyridoxal-5-phosphate)trans-1,2-cyclohexylenediamine

A 14 gm (0.02 mole) portion of the diimine product of Example 15 wasdissolved in 200 ml of 1:1 water:methanol. The resulting solution wassparged with argon, and 1.0 gm of 5% platinum on carbon was added. Thesystem was flushed with hydrogen. The hydrogen pressure was increased to50 psig for 16 hr at 25° C. The reaction product was filtered throughCELITE, and the resulting solution ofN,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclohexyldiamine or sodium5-(N-(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridyl)methylideneamino-trans-1,2-cyclohexyliminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphatewas concentrated in vacuo to about 20 ml and cooled to 0° C. to inducecrystallization. The product was isolated by filtration, washed withcold H₂ O and dried in vacuo. ¹ H NMR (D₂ O, 400 MHz) delta 7.45 (s,pyr-H), 4.53 (d, CH₂ OP, J_(HP) =4.9 Hz), 3.83 (dd, N-CH₂ -pyr), 2.72(br s, cyclo-(CH₂)₄ (CH)₂ (NH)₂ -), 1.88 (s, pyr-CH₃), 1.83-1.08 (3 brs, cyclo-(CH₂)₄ (CH)₂ (NH)₂ -).

EXAMPLE 18 N,N'-bis(pyridoxal-5-phosphate)-1,2-cyclo(alkylene orarylene)diamines

Repeating the procedure of Example 17 but replacing the diimine productof Example 15 with the diimine products prepared in accordance with theprocedure of Example 16 yields the corresponding diamines:

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclopentylenediamine,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cycloheptylenediamine,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclooctylenediamine,

N,n'-bis(pyridoxal-5-phosphate)-cis-1,2-cyclohexylenediamine,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,3-cyclohexylenediamine,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,4-cyclohexylenediamine,

N,N'-bis(pyridoxal-5-phosphate)-1,2-phenylenediamine, and

N,N'-bis(pyridoxal-5-phosphate)-cis-1,4-cyclohexylenediamine.

EXAMPLE 19N,N'-bis-(pyridoxal-5-phosphate)-trans-1,2-cyclohexylenediamine-N,N'-diaceticacid

The diamine from Example 17 was charged to a one liter 3-neck flask, andthe pH was adjusted to 11 with 5N NaOH. Then 5.6 gm (0.04 mole) ofbromoacetic acid was dissolved in 10 ml of water and added dropwise tothe stirred diamine solution while maintaining the pH at 11. Thereaction was warmed to 50° C. and stirred for 16 hr. 50 gm of weaklyacidic cation exchange resin (AMBERLITE IRC-50) was added, and the pHdropped to 6.7. The resin was removed by filtration, and 15 gm of cationexchange resin (DOWEX 50W-X8) was added. The pH dropped to 3.8.

The solution was filtered, and all of the solvent was evaporated fromthe filtrate to yield a foamy solid. The solid was dissolved in 30 ml of88% formic acid, and the product was precipitated by the addition of 150ml of methanol followed by 150 ml of ethanol. The solvent mixture wasdecanted from the gummy solid and discarded. The solid was dissolved ina minimum amount of deionized water (about 100 ml), and the product wasallowed to stand overnight at 25° C. The product was isolated byfiltration, washed with 50 ml of cold water, 25 ml of ethanol and thendried in vacuo to yield the product. The compound was recrystallized bythe same procedure to yieldN,N'-bis-(pyridoxal-5-phosphate)-trans-1,2-cyclohexylenediamine-N,N'-diaceticacid orN,N'-bis(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridylmethyl)-trans-1,2-cyclohexylenediamine-N,N'-diaceticacid (DPCP) with a melting point (decomposition) of 221°-226° C. ¹ H NMR(D₂ O, 400 MHz) delta 7.53 (s, pyr-H), 4.58 (d, CH₂ OP, J_(HP) =5.9 Hz),3.89 (dd, N-CH₂ -pyr), 3.31 (s, CH₂ COOH), 2.78 (br s, cyclo-(CH₂)₄(CH)₂ (NH)₂ -), 1.93 (s, pyr-CH₃), 1.90-1.15 (3 br s, cyclo-(CH₂)₄ (CH)₂(NH)₂ -).

EXAMPLE 20 N,N'-bis-(pyridoxal-5-phosphate)cyclo(alkylene andarylene)diamine-N,N'-diacetic acids

Repeating the procedure of Example 19 but replacing the diamine ofExample 17 with the diamines of Example 18 yields the corresponding:

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclopentylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cycloheptylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclooctylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-cis-1,2-cyclohexylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,3-cyclohexylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,4-cyclohexylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-phenylenediamine-N,N'-diacetic acid,and

N,N'-bis(pyridoxal-5-phosphate)-cis-1,4-cyclohexylenediamine-N,N'-diaceticacid.

EXAMPLE 21 Chelates

Repeating the procedure of Example 7 but replacingN,N'-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid withequimolar amounts of the products of chelate forming compounds producedin accordance with Examples 19 and 20 and using equimolar amounts of thesoluble chlorides, carbonates or nitrates of Mn⁺², Cr⁺³, Fe⁺², Fe⁺³,Co⁺³, Ni⁺², Cu⁺², Pr⁺³, Nd⁺³, Sm⁺³, Yb⁺³, Gd⁺³, Tb⁺³, Ho⁺³, or Er⁺³yields the sodium-calcium salts of the respective metal ion chelates of

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclohexylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclopentylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cycloheptylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclooctylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-cis-1,2-cyclohexylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,3-cyclohexylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,4-cyclohexylenediamine-N,N'-diaceticacid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-phenylenediamine-N,N'-diacetic acid,and

N,N'-bis(pyridoxal-5-phosphate)-cis-1,4-cyclohexylenediamine-N,N'-diaceticacid.

EXAMPLE 22 Pyridoxal-5-phosphate (N-methylethanolamine)monoester

2.04 gm (0.01 mole) of pyridoxal hydrochloride is dissolved in 50 ml ofdry THF containing 0.05 gm (0.02 mole) of sodium hydride with stirring.When gas evolution had ceased (about 15 min), 1.71 gm (0.01 mole) ofbenzyl bromide is added, and after stirring overnight, the solution isbrought to dryness in vacuo. The sticky solid is suspended in 50 ml ofdry methylene chloride and following addition of 3.0 gm (0.03 mole) oftriethylamine, the slurry is cooled to 0° C. 1.38 gm (0.01 mole) of2-chloro-3-methyl-1-oxa-3-aza-2-phosphacyclopentane (prepared by themethod of Jones, et al, J. Chem. Soc. Perkin trans I. p 199 (1985)) isadded with vigorous stirring. The suspension is stirred for 1 hr at rmtemp, and then 100 ml of water is added. The methylene chloride layer isseparated, dried over MgSO₄, and the solvent removed in vacuo. Additionof diethyl ether yields the intermediate product as a hydroscopic whitesolid (1.8 gm, 50% yield). The intermediate is oxidized with excessdinitrogen tetroxide in methylene chloride at -78° C., and then istreated with aqueous HCl in THF under reflux to give the(N-methylethanolamine)monoester of pyridoxal-5-phosphate in an overallyield of 40%.

EXAMPLE 23N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)ethylenediimine

Repeating the procedure of Example 1 but replacing pyridoxal-5-phosphatewith the product of Example 22 yieldsN,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)ethylenediimineor sodium5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)methylideneaminoethyleneiminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoricacid, N-methylethanolamine ester.

EXAMPLE 24 Other Monoester Diimines

Repeating the procedure of Example 23 with 1,3-diamino-n-propane,1,2-diamino-n-propane, 1,2-diaminoisopropane, 1,2-diamino-n-butane,1,4-diamino-n-butane, 1,3-diamino-n-butane, and1,2-diamino-3-methylpropane yields the correspondingN,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,3-(n-propylene)diimine,N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-(n-propylene)diimine,N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-isopropylenediimine,N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-(n-butylene)diimine,N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,4-(n-butylene)diimine,N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,3-(n-butylene)diimine,andN,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-(3-methyl)propylenediimine.

Repeating the procedure of Example 23 with trans-1,2-diaminocyclohexane,trans-1,2-diaminocyclopentane, trans-1,2-diaminocycloheptane,trans-1,2-diaminocyclooctane, o-aminoaniline, andcis-1,2-diaminocyclohexane, yields the correspondingN,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclohexylenediimine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclopentylenediimine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cycloheptylenediimine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclooctylenediimine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-phenylenediimine,and

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-cis-1,2-cyclohexylenediimine.

EXAMPLE 25N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)ethylenediamine

Repeating the procedure of Example 3 but substituting the diimineproduct of Example 23 for the diimine product of Example 1 yieldsN,N'-bis(pyridoxal-5-phosphate(N-methyl-ethanolamine)monoester)ethylenediamineor5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)methylaminoethyleneaminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoricacid, N-methyl-ethanolamine ester.

EXAMPLE 26 Other Monoester Diamines

Repeating the procedure of Example 25 but substituting the diimineproducts of Example 24 for the diimine product of Example 23 yields

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,3-(n-propylene)diamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-(n-propylene)diamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-isopropylenediamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-(n-butylene)diamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,4-(n-butylene)diamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,3-(n-butylene)diamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-(3-methyl)propylenediamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclohexylenediamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclopentylenediamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cycloheptylenediamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclooctylenediamine,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-phenylenediamine,and

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-cis-1,2-cyclohexylenediamine.

EXAMPLE 27 DPDP-phosphate monoester

Repeating the procedure of Examples 5 and 6 but replacing the diamine ofExample 3 with the product of Example 25 yieldsN,N'-bis(pyridoxal-5-phosphate-(N-methyl-ethanolamine)monoester)ethylenediamine-N,N'-diaceticacid, sodium salt or N-methylethanolamine phosphate ester of5-(N-(3-hydroxy-2-methyl-5-phosponomethyl-4-pyridyl)methylaminoethyleneaminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoricacid, sodium salt.

EXAMPLE 28 Other Diamine-N,N'-diacetic Acid Phosphate Monoesters

Repeating the procedure of Example 27 but replacing the products ofExample 26 for the product of Example 25 yields

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,3-(n-propylene)diamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)mono-ester)-1,2-(n-propylene)diamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-isopropylenediamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-(n-butylene)diamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,4-(n-butylene)diamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,3-(n-butylene)diamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-(3-methyl)propylenediamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclohexylenediamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclopentylenediamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cycloheptylenediamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-trans-1,2-cyclooctylenediamine-N,N'-diaceticacid salt,

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-1,2-phenylenediamine-N,N'-diaceticacid salt, and

N,N'-bis(pyridoxal-5-phosphate(N-methylethanolamine)monoester)-cis-1,2-cyclohexylenediamine-N,N'-diaceticacid salt.

EXAMPLE 29 Chelates

Repeating the procedure of Example 7 but replacingN,N'-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid withan equimolar amount of the chelate forming compound produced inaccordance with Example 27, yields the sodium-calcium Mn(II) chelate ofN,N'-bis(pyridoxal-5-phosphate-(N-methyl-ethanolamine)monoester)ethylenediamine-N,N'-diaceticacid.

Repeating this procedure, replacing manganese dichloride tetrahydratewith equimolar amounts of the soluble chlorides, carbonates or nitratesof Cr⁺³, Fe⁺², Fe⁺³, Co⁺³, Ni⁺², Cu⁺², Pr⁺³, Nd⁺³, Sm⁺³, Yb⁺³, Gd⁺³,Tb⁺³, Dy⁺³, Ho⁺³, or Er⁺³ yields the sodium-calcium salts of therespective metal ion chelates ofN,N'-bis(pyridoxal-5-phosphate-(N-methyl-ethanolamine)monoester)ethylenediamine-N,N'-diaceticacid.

EXAMPLE 30 Other Chelates

Repeating the procedures of Example 29 but replacing the products ofExample 28 for the product of Example 27 yields the Mn⁺², Cr⁺³, Fe⁺²,Fe⁺³, Co⁺³, Ni⁺², Cu⁺², Pr⁺³, Nd⁺³, Sm⁺³, Yb⁺³, Gd⁺³, Tb⁺³, Ho⁺³, orEr⁺³ ion chelates of the sodium-calcium salts of the diacetic acidchelating agents of Example 28.

EXAMPLE 31 DPDP-(mono)acetic Acid Analog Synthesis

10 gms (0.017 mole) of the diamine from Example 3 was dissolved in 25 mlof 1:1 water/methanol and charged to a 250 ml 4-neck flask equipped withtwo addition funnels, pH electrode, thermometer and stir bar. 1.4 gms(0.035 mole) of NaOH and 2.4 gm (0.017 mole) of bromoacetic acid wereeach dissolved in 10 ml of deionized water and charged to the twoaddition funnels. Sufficient NaOH was added to the stirring diaminesolution to bring the pH to about 11, which raised the temperature toabout 40° C. The temperature was maintained at 40° C., and bromoaceticacid and NaOH were added concurrently to maintain the pH at 11 over thecourse of 3 hr. The reaction was monitored by HPLC. Dowex 50W-X8 resinwas added to lower the pH from 11.1 to 3.1, the solution was filtered,and resin was washed with 100 ml of deionized water. The pH of thefiltrate was about 3.3. 5 ml of 97% formic acid was added, and the pHdropped to 3.0. Then 10 ml of isopropyl alcohol was added with a fewseed crystals, the product stirred overnight at 30° to 40° C., and thenallowed to cool to 25° C. The crude product was collected by filtrationand washed with deionized water. The crude product was then dried at 50°C. in vacuo to yield 3 gms of product (30% yield). The product can berecrystallized from a formic acid/water mixture to yield 2.4 gms in96-98% purity by HPLC to yieldN,N'-bis-(pyridoxal-5-phosphate)ethylenediamine-N-acetic acid.

EXAMPLE 32 Other (Mono)acetic Acids

Repeating the procedure of Example 31 with the products of Examples 4,17 and 18 yields the corresponding

N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-propylene)diamine-N-acetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-propylene)diamine-N-acetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-isopropylenediamine-N-acetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-(n-butylene)diamine-N-acetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,4-(n-butylene)diamine-N-acetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,3-(n-butylene)diamine-N-acetic acid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-(3-methyl)propylenediamine-N-aceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclohexylenediamine-N-aceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclopentylenediamine-N-aceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cycloheptylenediamine-N-aceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclooctylenediamine-N-aceticacid,

N,N'-bis(pyridoxal-5-phosphate)-cis-1,2-cyclohexylenediamine-N-aceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,3-cylohexylenediamine-N-aceticacid,

N,N'-bis(pyridoxal-5-phosphate)-trans-1,4-cyclohexylenediamine-N-aceticacid,

N,N'-bis(pyridoxal-5-phosphate)-1,2-phenylenediamine-N-acetic acid, and

N,N'-bis(pyridoxal-5-phosphate)-cis-1,4-cyclohexylenediamine-N-aceticacid.

EXAMPLE 33 Other Chelates

Repeating the procedures of Example 29 but replacing the products ofExample 32 for the product of Example 27 yields the Mn⁺², Cr⁺³, Fe⁺²,Fe⁺³, Co⁺³, Ni⁺², Cu⁺², Pr⁺³, Nd⁺³, Sm⁺³, Yb⁺³, Gd⁺³, Tb⁺³, Ho⁺³, orEr⁺³ ion chelates of the sodium-calcium salts of the monoacetic acidchelating agents of Example 32.

EXAMPLE 34 N-pyridoxal-N'-(pyridoxal-5-phosphate)ethylenediimine

A 25 gm (0.123 mole) quantity of pyridoxal hydrochloride is slurried in100 ml of methanol, and 4.88 gm (0.123 mole) of NaOH is added. When thesolution is homogeneous, it is added dropwise to 7.5 gm of1,2-diaminoethane in 100 ml of methanol with stirring. After 60 min, amethanol solution containing 32.7 gm (0.123 mole) ofpyridoxal-5-phosphate and 4.88 gm (0.123 mole) of NaOH is added withvigorous stirring. The unsymmetrical imine product,5-(N-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)methylideneaminoethyleneiminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoricacid or N-pyridoxal-N'-(pyridoxal-5-phosphate)-ethylenediimine, isstirred for 1 hr, and the product is isolated by filtration. The diimineis washed with methanol (2×50 ml) and diethyl ether (2×50 ml), and driedin vacuo.

EXAMPLE 35 N-pyridoxal-N'-(pyridoxal-5-phosphate)ethylenediamine

Repeating the procedure of Example 3 but substituting the diimineproduct of Example 34 for the diimine product of Example 1 yields thecorresponding diamine product,5-(N-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)methylaminoethyleneaminomethyl)-2-hydroxy-3-methyl-5-pyridylmethylphosphoricacid or N-pyridoxal-N'-(pyridoxal-5-phosphate)-ethylenediamine.

EXAMPLE 36 DPMP Synthesis

Repeating the procedures of Example 5 and 6 with the product of Example35 yields the correspondingN-pyridoxal-N'-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acidorN-(3-hydroxy-5-hydroxyomethyl-2-methyl-4-pyridylmethyl)-N'-(3-hydroxy-2-methyl-5-phosphonomethyl-4-pyridylmethyl)ethylenediamine-N,N'-diaceticacid (DPMP).

EXAMPLE 37 Other Chelates

Repeating the procedures of Example 29 but replacing the product ofExample 36 for the product of Example 27 yields the Mn⁺², Cr⁺³, Fe⁺²,Fe⁺³, Co⁺³, Ni⁺², Cu⁺², Pr⁺³, Nd⁺³, Sm⁺³, Yb⁺³, Gd⁺³, Tb⁺³, Ho⁺³, orEr⁺³ ion chelates of the sodium-calcium salts ofN-pyridoxal-N'-(pyridoxal-5-phosphate)ethylenediamine-N,N'-diaceticacid.

We claim:
 1. A metal ion chelate of a chelating compound of the formula:##STR9## wherein: R is hydrogen or ##STR10## R₁ is hydrogen or ##STR11##with the proviso that at least one of R and R₁ is other than hydrogen;R₅and R₆ are each, independently, hydroxy, alkoxy having from 1 to 18carbons, hydroxy-substituted alkoxy having from 1 to 18 carbons, amino,or alkylamido having from 1 to 10 carbons; R₃ is alkylene having from 1to 8 carbons, 1,2-cycloalkylene having from 5 to 8 carbons, or1,2-arylene having from 6 to 10 carbons; R₄ is hydrogen, hydroxymethyl,alkyl having from 1 to 6 carbons, or ##STR12## each R₇ is,independently, hydrogen, hydroxy-substituted alkyl having from 1 to 18carbons, or aminoalkyl having from 1 to 18 carbons; and the chelatedmetal ion is a paramagnetic ion of a metal having an atomic number offrom 21 to 29, inclusive, 42, 44, or from 58 to 70, inclusive, or aphysiologically biocompatible inorganic or organic salt of said metalion chelate.
 2. A metal ion chelate according to claim 1 wherein R andR₁ are each other than hydrogen, or a salt of said metal ion chelate. 3.A metal ion chelate according to claim 1 wherein each R₇ is hydrogen, ora salt of said metal ion chelate.
 4. A metal ion chelate according toclaim 1 wherein R and R₁ are each other than hydrogen, R₅ and R₆ areeach, independently, hydroxy, alkoxy having from 1 to 8 carbons,hydroxyethyl, dihydroxypropyl, amino, or alkylamido having from 1 to 8carbons, or a salt of said metal ion chelate.
 5. A metal ion chelateaccording to claim 4 wherein R₃ is alkylene having from 2 to 6 carbons,or a salt of said metal ion chelate.
 6. A metal ion chelate ofN,N'-bis(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid or asalt thereof, wherein the metal ion is as recited in claim 1, or a saltof said metal ion chelate.
 7. A metal ion chelate according to claim 4wherein R₃ is cyclohexyl, or a salt of said metal ion chelate.
 8. Ametal ion chelate ofN,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclohexyldiamine-N,N'-diaceticacid or a salt thereof, wherein the metal ion is as recited in claim 1,or a salt of said metal ion chelate.
 9. A metal ion chelate according toclaim 1 wherein the metal ion is divalent or trivalent, or a salt ofsaid metal ion chelate.
 10. A metal ion chelate according to claim 1wherein the metal ion is chromium (III), manganese (II), iron (III),iron (II), cobalt (II), nickel (II), copper (II), praseodymium (III),neodymium (III), samarium (III), ytterbium (III), gadolinium (III),terbium (III), dysprosium (III), holmium (III), or erbium (III), or asalt of said metal ion chelate.
 11. A metal ion chelate according toclaim 1 wherein the metal ion is manganese (II), or a salt of said metalion chelate.
 12. A manganese (II) chelate ofN,N'-bis(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid or asalt thereof, or a salt of said manganese (II) chelate.
 13. A manganese(II) chelate ofN,N'-bis(pyridoxal-5-phosphate)-trans-1,2-cyclohexyldiamine-N,N'-diaceticacid or a salt thereof, or a salt of said manganese (II) chelate.
 14. Ametal ion chelate according to claim 1 in the form of a calcium salt ofan anionic chelate complex of a paramagnetic metal ion and a chelatingcompound of formula I, or a salt of said metal ion chelate.
 15. A metalion chelate or salt according to claim 14 wherein the molar ratio ofcalcium to chelating compound is from 0.05 to 1.0.
 16. A metal ionchelate in the form of a calcium salt of the manganese (II) chelate ofclaim 12 or a salt thereof.
 17. A metal ion chelate or salt according toclaim 16 wherein the molar ratio of calcium to chelating compound isfrom 0.05 to 1.0.
 18. A sodium-calcium salt of a manganese (II) chelateof N,N'-bis(pyridoxal-5-phosphate)ethylenediamine-N,N'-diacetic acid ora salt thereof.